Increasing evidence indicates that in eukaryotes cell growth (mass accumulation) is finely regulated in response to environmental and developmental conditions and can be deranged in human diseases such as cancer and diabetes (reviewed by Dixon and Fordham-Skelton, Curr. Opin. Plant Biol. 1:1 (1998); Johnston and Gallant, Bioessays 24: 54-64 (2002); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615-75 (2001); Kozma and Thomas, Bioessays 24:65-71 (2002); Schmelzle and Hall, Cell 103:253-62 (2000)). The rate of mass accumulation is controlled not simply by the availability of nutrients, but by signaling pathways that coordinate the activity of the cell growth machinery with nutritional, hormonal and mitogenic signals. Studies into the mechanism of action of rapamycin, an immunosuppressive and anti-cancer drug, led to the discovery of an evolutionarily conserved regulator of cell growth, the TOR (Target of Rapamycin) pathway (Brown et al., Nature 369:756-758 (1994); Chiu et al., Proc. Natl. Acad. Sci. USA 91:12574-12578 (1994); Kunz et al., Cell 73:585-596 (1993); Oldham et al., Genes Dev 14:2689-94 (2000); Sabatini et al., Cell 78:35-43 (1994); Sabers et al., J. Biol. Chem. 270:815-822. (1995); Zhang et al., Genes Dev 14:2712-24 (2000)). The complex of rapamycin with its receptor, FKBP12, binds directly to TOR and perturbs its function in a poorly understood fashion (Brown et al., Nature 377:441-446 (1995); Burnett et al., PNAS 95:1432-1437 (1998); Peterson et al., Biol Chem 275:7416-23 (2000); Zheng et al., Cell 82:121-130 (1995)). TOR is a member of the PIK-related family of proteins (Keith and Schreiber, Science 270:50-5 (1995)) that share homology with the catalytic domain of phosphatidylinositol 3-kinase (PI-3K), but appear to function as serine/threonine rather than lipid kinases. Studies in several organisms have shown that the TOR pathway regulates a variety of processes contributing to cell growth, including initiation of mRNA translation, ribosome synthesis, expression of metabolism-related genes, autophagy and cytoskeletal reorganization (recently reviewed by Schmelzle and Hall, Cell 103:253-62 (2000) and by Gingras et al., Genes Dev 15:807-26 (2001)). By interfering with the function of mammalian TOR, rapamycin inhibits progression through the G1 phase of the cell cycle in various cell types. Because of these anti-proliferative effects, rapamycin is a clinically valuable drug that is currently used to block immune rejection of transplanted organs (Saunders et al., Kidney Int. 59:3-16 (2001)) and in trials for the treatment of cancer (Dudkin et al., Clin. Cancer Res 7:1758-64 (2001); Hidalgo and Rowinsky, Oncogene 19, 6680-6 (2000)) and for the prevention of restenosis after angioplasty (Sousa et al., Circulation 104:2007-11 (2001)).
Mammalian TOR, mTOR (also known as RAFT1 or FRAP), phosphorylates at least two regulators of protein synthesis: S6K1 (formerly called p70 ribosomal S6 kinase) and an inhibitor of translation initiation, the eIF-4E binding protein 1 (4E-BP1) (Brunn et al., Science 277, 99-101 (1997); Burnett et al., PNAS 95:1432-1437 (1998); Hara et al., J. Biol. Chem. 272, 26457-63 (1997); Isotani et al., J Biol Chem 274:34493-8 (1999)). In mammalian cells, amino-acid deprivation leads to the dephosphorylation of both S6K1 and 4E-BP1 and to decreased rates of protein synthesis, effects that are rapidly reversed by the re-addition of amino acids (Fox et al., American Journal of Physiology Cell Physiology 274:43-1 (1998); Hara et al., J Biol Chem 273:14484-94 (1998)). Among amino acids, changes in leucine levels alone are sufficient to regulate the phosphorylation state and activity of both downstream components of the mTOR pathway (Hara et al., J Biol Chem 273:14484-94 (1998); Lynch et al., J. Cell Biochem. 77:234-51 (2000)). In addition to amino acid levels, mitochondrial function (Xu et al., Diabetes 50:353-60 (2001)), glycolysis (Dennis et al., Science 294:1102-5 (2001)), and cell stress (Parrott and Templeton, J. Biol. Chem. 274:24731-6. (1999)) regulate S6K1, as do growth factors, such as insulin (Lawrence and Brunn, Prog. Mol. Subcell. Biol. 26:1-31 (2001)).
Despite extensive efforts, how nutrients regulate the mTOR signaling pathway remains poorly understood. In particular, stimuli that activate (e.g. amino acids) or inhibit (e.g. mitochondrial uncouplers) downstream effectors of mTOR, such as S6K1 and 4E-BP1, fail to change the in vitro kinase activity of mTOR (Dennis et al., Science 294:1102-5 (2001); Hara et al., J. Biol. Chem. 272:26457-63 (1997)).
Thus, a greater understanding of mTOR/RAFT1/FRAP, a central component of a signaling pathway that modulates cell growth in response to nutrients and hormones and which is the target of the immunosuppressive drug rapamycin, would be useful in the diagnosis and treatment of cell growth disorders such as cancer.